Intestinal barrier dysfunction contributes to progression of gastrointestinal and systemic disease. Over the current and previous cycle of this award we have i) made the fundamental discovery that the tight junction complex undergoes continuous remodeling at steady state; ii) demonstrated that ABR (actin binding region) - mediated ZO-1 anchoring at the tight junction is reduced by physiological and pathological stimuli that increase paracellular permeability, i.e. reduce barrier function; iii) identified requsite roles for both ZO-1 and its binding partner occludin in TNF-induced increases in paracellular macromolecular (leak pathway) flux; iv) demonstrated that ZO-1 is an essential regulator of the occludin endocytosis that is necessary for TNF-induced barrier loss; v) discovered that ZO-1 acts as a scaffold linking occludin to claudin-2 thereby inhibiting flux across claudin-2 pores; vi shown that assembly of the occludin:ZO-1:claudin-2 complex is sufficient to reverse IL-13-induced increases in paracellular ion (pore pathway) flux; and vii) revealed an unexpected, essential role for ZO-1 in early epithelial polarization and 3D lumen organization. These advances, along with additional data, indicate that ZO-1 is central to regulation of diverse aspects of epithelial function. This is not surprising, as ZO-1 contains multiple protein-binding domains. Nevertheless, our understanding of the protein interactions mediated by ZO-1, their underlying molecular basis, and how they regulate epithelial function remains rudimentary. This fundamental knowledge gap limits our ability to develop viable therapeutic agents. This proposal seeks to bridge that gap using an integrated approach that builds on our strong published and preliminary data, exploits our rich toolbox which includes novel cell lines, stem cell cultures, an genetically- modified mice, and takes advantage of cutting-edge technologies. Specifically, we propose to define the molecular mechanisms by which ZO-1 regulates epithelial morphogenesis, homeostasis, and repair; determine the elements and interactions by which ZO-1 regulates leak pathway permeability and the impact of these on colitis; and determine the structural underpinnings of ZO-1-mediated pore pathway regulation. The detailed view of molecular interactions responsible for multiple ZO-1 functions and their impact on disease will provide new insights into the function of this evolutionarily ancient protein. The proposal is innovative because it will use cutting-edge approaches to define novel regulatory processes, elevate our understanding, and make it possible to develop means to restore diverse epithelial functions for therapeutic benefit. The proposed research is significant because it will identify specific molecular mechanisms and link these directly to disease progression. Finally, in addition to creating foundational knowledge necessary for development of intestinal epithelial-targeted therapies, the concepts, tools, and technologies generated will enable advances in understanding of epithelial diseases of many organs, as they are expected to be widely applicable for restoration of epithelial function.